Development of Real-time RT-PCR Assays for Detection and Quantification of Pineapple Mealybug Wilt-associated Viruses-1

Abstract

Pineapple mealybug wilt-associated virus-1 (PMWaV-1) is a major causative agent for pineapple disease. A real-time, one-step reverse transcription (RT)–polymerase chain reaction (PCR) assay using TaqMan probes was developed for the detection of PMWaV-1. Real-time RT-PCR, targeting the amplification of heat-shock protein 70 homolog (hsp70 h) gene fragment, was highly specific and capable of distinguishing PMWaV-1 from PMWaV-2 and PMWaV-3, which are two other common virus species in pineapple plants. Sensitivity assay revealed that real-time RT-PCR was approximately 100- to 1000-fold more sensitive than standard one-step RT-PCR, when total RNA of infected plant and PMWaV-1 hsp70 h transcript were used, respectively. This real-time RT-PCR assay is rapid, specific and sensitive, which can be used for PMWaV-1 diagnosis as well as surveillance.